Fluoroquinolones are antimicrobial agents used frequently around the world. Fluoroquinolone-resistant and multidrug-resistant gram-negative bacterial pathogens are emerging infectious disease agents of worldwide concern. The long-term goals of the Zechiedrich laboratory are to determine how gram-negative bacteria respond to and develop resistance to antimicrobial agents and to use this knowledge to improve diagnosis of antibiotic-resistant bacterial infections, to prolong the usefulness of current drugs and to aid in the design of new therapeutic protocols. In the previous funding periods, we built a database that combines patient, hospital, and microbiological data from bacterial isolates that we have collected since 1999. Utilizing this database, we are working toward repurposing an off-patent antifungal drug as an antimicrobial agent with excellent activity against gram-negative pathogens. To discover new fluoroquinolone resistance mechanisms, we used a novel genome pooling, sequencing, and SNP analysis approach. In addition to uncovering the well- known gyrA target gene SNP, we discovered three novel SNPs that were present in every fluoroquinolone- resistant isolate in our collection. We also found 35 novel SNPs associated with a fluoroquinolone susceptible phenotype and 287 SNPs associated with various additional antimicrobial resistance phenotypes. The goals of this competing renewal proposal are to determine how these genetic variants affect antibiotic resistance and to test the predictive power of these SNPs for reporting antibiotic resistance phenotypes. Success of this latter goal could be used in a rapid genome-based diagnostic assay of antibiotic resistance. Our team, at Baylor College of Medicine in the Texas Medical Center in Houston, Texas, is composed of molecular microbiologist, Zechiedrich (P.I.), physician scientist, Hamill (co-I), genomics expert, Sucgang (co-I), computational biologist, Barth (co-I), and crystallographer, Lee (co-I). Together we will achieve these goals with the following specific aims: 1. Determine how newly discovered SNPs affect antibiotic resistance; 2. Identify and characterize genetic alterations associated with fluoroquinolone resistance and additional specific antibiotic resistance phenotypes; and 3. Test the diagnostic power of SNP detection as a reporter of antibiotic resistance.